This invention relates to a method for the purification of a substance capable of proliferating intestinal bacteria belonging to the genus Bifidobacterium, i.e. bifidobacteria, and more detailedly relates to a method for the purification of a bifidobacteria-proliferating substance in soybean whey.
Intestinal bifidobacteria are generally regarded as phsiologically preferred for infants to adults. Particularly, they are said to have effects of the prevention of infection in the intestinal tract, the reinforcement of immune function, the inhibition of putrefaction in the intestine, the decomposition of carcinogens, the production of vitamins, etc.
In order to exert such effects, it is important to make an environment in the intestine such that bifidobacteria take root and proliferate, and as a method therefor it has been tried to maintain the number of the bifidobacteria in the intestine to a high level by orally administering a substance promoting the proliferation of bifidobacteria solely or together with a Bifidobacterium culture.
Heretofore, oligosaccharides contained in soybeans, such as stachyose and raffinose, are known as saccharides proliferating bifidobacteria. However, the effect of bifidobacteria proliferation of these saccharides is inferior to that of the bifidobacteria-proliferating substance contained in soybean milk.
As for bifidobacteria-proliferating substances contained in soybean milk, it is disclosed, for example in Japanese Published Patent Application Nos. 142566/1976 and 85390/1980, etc. that soybean milk is effective for the growth of bifidobacteria. However, these publications do not disclose which ingredients are effective.
Further, processes for the preparation of a bifidobacteria proliferation-promoting substance from soybean milk (an extract of defatted soybean with water) are reported in Japanese Published Patent Application Nos. 179064/1984, 66978/1985 etc. and the processes comprise a protein removal step wherein phosphoric acid or hydrochloric acid is added to soybean milk, a neutralization step wherein calcium hydroxide is used in the presence of calcium chloride, a heating and precipitate separation step and a desalting and concentration step (reverse osmotic pressure treatment).
However, in the heating step (100.degree. C., 10 minutes) in the preparation processes there is a problem that the amino acids and the reducing saccharides in the soybean whey react. Further, the liquid at the stage when the heating and precipitate separation step was completed contains various impurities, and when the liquid is directly subjected to reverse osmotic pressure treatment, the reverse osmosis membrane is apt to be fouled and clogged and permeate flux decreases. Further, the purification degree of the reverse osmotic pressure-treated liquid is insufficient (small amounts of colored substances, salts, proteins, etc. are contained) and the use thereof is limited.
On the other hand, the present inventors proposed a process for the production of a bifidobacteria-proliferating substance which comprises extracting defatted soybeans, as the raw material, with an aqueous alcohol solution and removing the alcohol. Further, the present inventors invented as a method including the improvement of this process a method for the purification of a bifidobacteria-proliferating substance which comprises ultrafiltering soybean whey, subjecting the permeate to active carbon treatment and electrodialyzing the liquid after the removal of the active carbon. This method was filed as U.S. patent application Ser. No. 07/537,664 (European Patent Application No. 90306429.3/1990). In this method an aqueous solution of a bifidobacteria-proliferating substance is obtained as an electrodialysis-treated liquid by carrying out the ultrafiltration using an ultrafiltration membrane having a fractional molecular weight of 20,000 to 100,000 to remove high molecular substances, mainly proteins having a molecular weight equal to or more than the fractional molecular weight, subjecting the permeate to active carbon treatment to remove low molecular proteins, particularly 2 S proteins (S:Svedbery unit; a unit of sedimentation coefficient) becoming a cause of membrane fouling in the succeeding electrodialysis, colored materials, etc., and electrodialyzing the liquid after the removal of the active carbon to remove salts contained in a small amount, and subjecting the electrodialyzed liquid to ion exchange resin treatment to obtain an aqueous solution of a bifidobacteria-proliferating substance. This ion exchange resin-treated liquid is substantially free of proteins and is usually colorless and transparent, and therefore a product of high completion degree such that it can be applied as a bifidobacteria-proliferating substance as such, or as syrup after concentration or as a powder after successive drying.
However depending on soybean whey to be used, there sometimes arises a case where when the concentration factor of the liquid to be treated is raised in the ultrafiltration step in the method of U.S. patent application Ser. No. 07/537,664 (European Patent Application No. 90306429.3) in order to enhance the amount of the bifidobacteria-proliferating substance transferred to the permeate up to an economically paying basis, permeate flux greatly decreases. Namely, there is a case where when such a concentration factor as paying economically is tried to be attained, it takes a very long time for the treatment.
In this connection, the above U.S. patent application Ser. No. 07/537,664 (European Patent Application No. 90306429.3) has no position of prior art in judgment of the unobviousness (inventive step) of the invention of this application.